Bottom Line:
Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II.Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase.Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20.

ABSTRACTIn Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

fig6: The effect of Cdc20-GR and -4AV injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with Cdc20-GR or -4AV were treated as in Fig. 3 A. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of control (left), or Cdc20-GR injected (middle), or Cdc20-4AV injected (right) oocytes. Oocytes were treated as in Fig. 4 B. Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus. (C) The localization of Cdc20 is affected by checkpoint protein mutants. Cdc20-YFP and histone H2B-RFP mRNA and Bub1dn- or Mad2ΔC- or BubR1d-injected oocytes were treated as above, and fixed 5–6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). The histone H2B-RFP and Cdc20-YFP signals are shown individually, as well as overlaid (merge). Note the dotted appearance of the Cdc20-YFP signal around the chromosomes, suggesting a kinetochore localization, in the Mad2ΔC coinjected oocytes, which is weaker in the BubR1d coinjected, and seems absent from the Bub1dn coinjected oocytes. (D) The localization of the Cdc20-GR or -4AV YFP-fusion proteins. Cdc20-GR-YFP– or Cdc20-4AV-YFP– and H2B-RFP–injected oocytes were treated as above, and fixed 6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). Note the dotted appearance of the Cdc20-4AV-YFP signal around the chromosomes, suggesting a kinetochore localization, which is not detectable in Cdc20-GR-YFP–injected oocytes. Bars: (B–D) 10 μm.

Mentions:
As shown in Fig. 6, the Cdc20-GR mutant caused early anaphase in the first meiosis (Fig. 6 A; Table I), but could not force exit from metaphase II (Fig. 6 B; Table II). This mutant, thus, behaved similarly to the above tested dominant-negative checkpoint proteins, and could, despite its checkpoint resistance, not activate the APC/C to induce exit from meiosis II.

fig6: The effect of Cdc20-GR and -4AV injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with Cdc20-GR or -4AV were treated as in Fig. 3 A. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of control (left), or Cdc20-GR injected (middle), or Cdc20-4AV injected (right) oocytes. Oocytes were treated as in Fig. 4 B. Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus. (C) The localization of Cdc20 is affected by checkpoint protein mutants. Cdc20-YFP and histone H2B-RFP mRNA and Bub1dn- or Mad2ΔC- or BubR1d-injected oocytes were treated as above, and fixed 5–6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). The histone H2B-RFP and Cdc20-YFP signals are shown individually, as well as overlaid (merge). Note the dotted appearance of the Cdc20-YFP signal around the chromosomes, suggesting a kinetochore localization, in the Mad2ΔC coinjected oocytes, which is weaker in the BubR1d coinjected, and seems absent from the Bub1dn coinjected oocytes. (D) The localization of the Cdc20-GR or -4AV YFP-fusion proteins. Cdc20-GR-YFP– or Cdc20-4AV-YFP– and H2B-RFP–injected oocytes were treated as above, and fixed 6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). Note the dotted appearance of the Cdc20-4AV-YFP signal around the chromosomes, suggesting a kinetochore localization, which is not detectable in Cdc20-GR-YFP–injected oocytes. Bars: (B–D) 10 μm.

Mentions:
As shown in Fig. 6, the Cdc20-GR mutant caused early anaphase in the first meiosis (Fig. 6 A; Table I), but could not force exit from metaphase II (Fig. 6 B; Table II). This mutant, thus, behaved similarly to the above tested dominant-negative checkpoint proteins, and could, despite its checkpoint resistance, not activate the APC/C to induce exit from meiosis II.

Bottom Line:
Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II.Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase.Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20.

ABSTRACTIn Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.